THE NA+ H+ EXCHANGER ISOFORM-1 (NHE1) IS A NOVEL MEMBER OF THE CALMODULIN-BINDING PROTEINS - IDENTIFICATION AND CHARACTERIZATION OF CALMODULIN-BINDING SITES/

The Na+/H+ exchange activity (NHE1 human isoform) is rapidly activated in response to growth factors and hyperosmotic stress. To get insight into the mechanism of NHE1 activation, we studied the direct interact ion of a ubiquitous Ca2+-dependent regulatory factor, calmodulin (CaM) with NHE1. Binding experiments with CaM-Sepharose, as well as fluores cence measurements with dansylated CaM, revealed that the NHE1 cytopla smic domain strongly binds CaM in a Ca2+-dependent manner. Fusion prot ein analysis with deletion mutants provided evidence for high (K-d sim ilar to 20 nM) and intermediate (K-d similar to 350 nM) affinity CaM-b inding sites located in neighboring regions of NHE1 (amino acids 636-6 56 and 657-700). To assess a regulatory role of CaM-binding sites, sev eral cDNAs having deletion and point mutations in the high affinity si te were generated and expressed in the exchanger-deficient fibroblast cell line PS120. Deletion and point mutations of positively charged re sidues of the high affinity CaM-binding site resulted in up to 50 and 80% reductions of cytoplasmic alkalinization caused by growth factors (alpha-thrombin, etc.) and 100 mM sucrose, respectively. In these muta nts, the reduction in alkalinization was apparently in proportion to t hat of the CaM-binding ability. These results suggest that binding of Ca2+/CaM to the high affinity site is involved at least partly in the activation of NHE1 in response to different extracellular signals.